Low-grade PanINs Research Articles

Genome-Wide Somatic Copy Number Alterations and Mutations in High-Grade Pancreatic Intraepithelial Neoplasia

To obtain a better understanding of the genetic alterations of high-grade pancreatic intraepithelial neoplasia (HG-PanIN), we performed whole-genome copy number analysis by using single nucleotide polymorphism microarrays and targeted next-generation sequencing of 11 microdissected HG-PanIN and two low-grade PanIN lesions associated with HG-PanIN. HG-PanIN mutation profiles were compared with those of their associated invasive pancreatic ductal adenocarcinoma. All PanIN lesions harbored somatic KRAS mutations. The most common copy number losses in the HG-PanIN were at the CDKN2A (9p21), TP53 (17p13), and SMAD4 (18q21) loci. Chromosomal losses in HG-PanIN were also found at 6p25-p24, 6q11-q27, 12q24, and 17q23-q24. Biallelic inactivation of CDKN2A and TP53 was detected in five of eight and in three of eight evaluable PanIN lesions, respectively. None of the HG-PanIN lesions had SMAD4 mutations or homozygous deletion. Copy number gains were noted at the MYC (8q24) and CCNE1 (19q12) loci and at 1q25-q31. Four HG-PanINs and one low-grade PanIN harbored chromothripsis-like regions. Five of seven pancreatic ductal adenocarcinomas evaluated had additional mutations that were not found in their associated HG-PanIN. HG-PanIN harbors widespread copy number alterations and commonly shows evidence of biallelic inactivation of CDKN2A and TP53 but not SMAD4. Chromothripsis events contribute to the copy number alterations of HG-PanIN.

IL2RG, identified as overexpressed by RNA-seq profiling of pancreatic intraepithelial neoplasia, mediates pancreatic cancer growth.

Pancreatic ductal adenocarcinoma evolves from precursor lesions, the most common of which is pancreatic intraepithelial neoplasia (PanIN). We performed RNA-sequencing analysis of laser capture microdissected PanINs and normal pancreatic duct cells to identify differentially expressed genes between PanINs and normal pancreatic duct, and between low-grade and high-grade PanINs. One of the most highly overexpressed transcripts identified in PanIN is interleukin-2 receptor subunit gamma (IL2RG) encoding the common gamma chain, IL2Rγ. CRISPR-mediated knockout of IL2RG in orthotopically implanted pancreatic cancer cells resulted in attenuated tumor growth in mice and reduced JAK3 expression in orthotopic tumors. These results indicate that IL2Rγ/JAK3 signaling contributes to pancreatic cancer cell growth in vivo.

Abstract 1857: Early detection of pancreatic intraepithelial neoplasms (PanINs) in transgenic mouse model by hyperpolarized 13C metabolic magnetic resonance imaging

Abstract Pancreatic cancer, one of the most lethal solid tumors, is an aggressive disease that develops relatively symptom-free. Pre-invasive pancreatic intraepithelial neoplasias (PanINs) have been identified as precursor lesions to pancreatic cancer. While there is growing evidence supporting PanIN's genetic links to pancreatic cancer, there is no non-invasive method to detect them. There is an unmet need of novel strategies for detection of pancreatic cancer at the earliest stages, preferably at the stage of PanINs. Hyperpolarized magnetic resonance imaging (HP-MRI) has shown potential for early detection of cancers and monitoring therapeutic efficacy. Here we have employed in-vivo 13C pyruvate metabolic imaging and ex-vivo nuclear magnetic resonance (1H-NMR) metabolomics to identify and understand metabolic changes, to enable detection of early PanINs and its progression to advanced PanINs and pancreatic cancer. Genetically engineered mouse (GEM) models with progression of PanIN lesions and control animals, with no pancreatic lesions, were employed in this study. Hyperpolarization of pyruvate and in-vivo 13C MRS were performed using Hypersense (Oxford Instruments) and 7T MRI scanner with a dual tuned 1H/13C volume coil respectively. Tissue alanine and lactate concentrations were determined using a Bruker 500 MHz NMR spectrometer coupled with cryo-probe. Histology and immunohistochemistry were performed on excised tissue samples. P48Cre mice were used as controls and P48Cre;LSLKrasG12D mice were used for detection of early and advanced PanINs, which usually develop in these mice between 12 and 20 months. The imaging experiments were performed at the different stages of the disease. Progression of disease from tissue containing predominantly low-grade PanINs to tissue with high-grade PanINs showed a decreasing alanine/lactate concentration ratio. Real time in-vivo 13C MRS was used to measure non-invasively changes of alanine and lactate metabolites with disease progression and in control mice, following injection of hyperpolarized pyruvate. The alanine-to-lactate (Ala/Lac) signal intensity ratio was found decreased as the disease progressed from low-grade PanINs to high-grade PanINs. These results demonstrate that there are significant alterations of alanine transaminase (ALT) and lactate dehydrogenase (LDH) activities which favor the transformation of aggressive pancreatic cancer from PanINs lesion. Our results suggest that real-time conversion kinetic rate constants kPA(pyruvate-to-alanine) and kPL(pyruvate-to-lactate) can be used as metabolic imaging biomarkers for assessing the early stage of pancreatic diseases. Findings from this highly promising HP-MRI technique could be rapidly translated to the clinic for early detection of pancreatic cancer in patients at high risk for developing the disease. Citation Format: Prasanta Dutta, Yu Zhang, Michelle Zoltan, Marilina Mascaro, Erick Riquelme, Jaehyuk Lee, Anirban Maitra, Florencia McAllister, Pratip Bhattacharya. Early detection of pancreatic intraepithelial neoplasms (PanINs) in transgenic mouse model by hyperpolarized 13C metabolic magnetic resonance imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1857. doi:10.1158/1538-7445.AM2017-1857

Abstract A113: The pattern of hMENA isoforms is regulated by TGF-β1 in pancreatic cancer and may predict patient outcome

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with the worst survival rate among solid cancers. The pressing needs for extending life expectancy of patients are the identification of early prognostic markers and novel druggable pathways. PDAC arises generally from pancreatic intraepithelial neoplasia (PanIN) and a dynamic interactions between tumor, stromal cells and autocrine and paracrine signaling lead to epithelial to mesenchymal transition (EMT), an early process in the natural history of pancreatic cancer. Cytoskeletal reorganization, extracellular matrix (ECM) remodeling, and matrix metalloproteinases (MMPs) contribute to PDAC aggressiveness in cooperation with soluble growth factors or cytokines, with TGF-β1 as crucial player. hMENA is an actin regulatory protein whose splicing program, mediated by the epithelial splicing regulatory proteins (ESRPs), has been associated with the EMT process. Our previous studies indicated that alternative splicing of hMENA, generates hMENA11a and hMENAΔv6 isoforms with opposite roles in cell proliferation and invasion in breast and lung cancers. Alternative splicing is known to play a prominent role in tumor progression and tumorigenesis and the derived isoforms may represent powerful diagnostic and prognostic factors as we have recently shown for hMENA alternative splicing in early stage non-small cell lung cancer (NSCLC). The aim of this study is to investigate the role of TGF-β1 on the expression and function of hMENA isoforms in PDAC, and verify whether the expression pattern of hMENA isoforms may impact patient outcome. Methods: We analyzed the expression pattern of hMENA isoforms by immunohistochemistry, using anti-pan hMENA and specific anti-hMENA11a antibodies, in 285 PDACs, 15 PanINs, 10 pancreatitis, and normal pancreas, evaluating the patient outcome. The functional role of hMENA isoforms were analyzed by loss and gain of function experiments in untreated and TGF-β1-treated PDAC cell lines. Results: In a panel of pancreatic cancer cell lines, hMENA11a expression correlates with an epithelial phenotype, while hMENAΔv6 expression with a mesechymal phenotype, with low E-cadherin and high vimentin expression. hMENA11a knock-down in PDAC cell lines affected cell-cell adhesion but not cell invasion. TGF-β1 cooperated with β-catenin signalling to up-regulate hMENA and hMENAΔv6 expression but not hMENA11a. The hMENA/hMENAΔv6 up-regulation play a crucial role in cell invasiveness and in TGF-β1-induced EMT. After TGF-β1 treatment, hMENA/hMENAΔv6 were mobilized from focal adhesion to actin stress fibers, and the silencing of these isoforms significantly inhibited the TGF-β1-induced EMT in PANC-1. Functionally, in the absence of hMENA11a, the hMENA/hMENAΔv6 up-regulationis crucial for SMAD2-mediated TGF-β1 signalling, migration, invasion and MMPs activities. Pan hMENA immunostaining, absent in normal pancreas and low-grade PanINs, was weak in PanIN-3 and had higher levels in virtually all PDACs with 64% of cases showing strong staining. Conversely, the anti-invasive hMENA11a isoform only showed strong staining in 26% of PDAC. The absence of hMENA11a in a subset (34%) of pan-hMENA-positive tumors significantly correlated with poor outcome, in agreement with experimental results. Conclusions: hMENA isoforms are regulated differently by TGF-β1, and the pattern of expression of hMENA isoforms is crucial in TGF-β1-dependent EMT and cell invasion. The pattern of expression of hMENA isoforms correlates with PDAC patient outcome and it could be used in specific clinical settings for the choice of the most effective treatment of PDAC patients. Our data provide new insights into molecular pathways involved in PDAC biology and suggest that hMENA-related pathways are promising targets for the development of new prognostic and therapeutic tools in PDAC. Citation Format: Roberta Melchionna, Pierluigi Iapicca, Francesca Di Modugno, Paola Trono, Isabella Sperduti, Matteo Fassan, Ivana Cataldo, Borislav C. Rusev, Rita T. Lawlor, Maria Grazia Diodoro, Michele Milella, Gian Luca Grazi, Mina J. Bissell, Aldo Scarpa, Paola Nisticò. The pattern of hMENA isoforms is regulated by TGF-β1 in pancreatic cancer and may predict patient outcome [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A113.

The pattern of hMENA isoforms is regulated by TGF-β1 in pancreatic cancer and may predict patient outcome

ABSTRACTPancreatic ductal adenocarcinoma (PDAC) is an aggressive disease in need of prognostic markers to address therapeutic choices. We have previously shown that alternative splicing of the actin regulator, hMENA, generates hMENA11a, and hMENAΔv6 isoforms with opposite roles in cell invasion. We examined the expression pattern of hMENA isoforms by immunohistochemistry, using anti-pan hMENA and specific anti-hMENA11a antibodies, in 285 PDACs, 15 PanINs, 10 pancreatitis, and normal pancreas. Pan hMENA immunostaining, absent in normal pancreas and low-grade PanINs, was weak in PanIN-3 and had higher levels in virtually all PDACs with 64% of cases showing strong staining. Conversely, the anti-invasive hMENA11a isoform only showed strong staining in 26% of PDAC. The absence of hMENA11a in a subset (34%) of pan-hMENA-positive tumors significantly correlated with poor outcome. The functional effects of hMENA isoforms were analyzed by loss and gain of function experiments in TGF-β1-treated PDAC cell lines. hMENA11a knock-down in PDAC cell lines affected cell–cell adhesion but not invasion. TGF-β1 cooperated with β-catenin signaling to upregulate hMENA and hMENAΔv6 expression but not hMENA11a In the absence of hMENA11a, the hMENA/hMENAΔv6 up-regulation is crucial for SMAD2-mediated TGF-β1 signaling and TGF-β1-induced EMT. Since the hMENA isoform expression pattern correlates with patient outcome, the data suggest that hMENA splicing and related pathways are novel key players in pancreatic tumor microenvironment and may represent promising targets for the development of new prognostic and therapeutic tools in PDAC.

PanIN-associated pericyte, glial, and islet remodeling in mice revealed by 3D pancreatic duct lesion histology.

Pericytes and glial cells are accessory cells of neurovascular networks, which have been reported to participate in scar formation after tissue injury. However, it remains unclear whether similar reactive cellular responses occur in pancreatic intraepithelial neoplasia (PanIN). In this study we developed three-dimensional (3D) duct lesion histology to investigate PanIN and the associated pericyte, glial, and islet remodeling. Transparent mouse pancreata with a Kras(G12D) mutation were used to develop 3D duct lesion histology. Deep-tissue, tile-scanning microscopy was performed to generate panoramic views of the diseased pancreas for global examination of early stage and advanced duct lesion formation. Fluorescence signals of ductal and neurovascular networks were simultaneously detected to reveal associated remodeling. Significantly, in Kras(G12D)-mutant mice, when the low-grade PanINs emerge, duct lesions appear as epithelial buds with perilesional pericyte and glial activation. When PanINs occur in large scale (induced by cerulein injections to the mutant mice), the 3D image data identifies 1) aggregation of PanINs in clusters in space; 2) overexpression of the pericyte marker NG2 in the PanIN microenvironment; and 3) epithelial in-growth to islets, forming the PanIN-islet complexes. Particularly, the PanIN-islet complexes associate with proliferating epithelial and stromal cells and receive substantial neurovascular supplies, making them landmarks in the atrophic lobe. Overall, perilesional pericyte and glial activation and formation of the PanIN-islet complex underline cellular heterogeneity in the duct lesion microenvironment. The results also illustrate the advantage of using 3D histology to reveal previously unknown details of neurovascular and endocrine links to the disease.

Clinical profile of PanIN lesions in tropical chronic pancreatitis.

Chronic pancreatitis (CP) found in the tropical countries is known to have a higher risk for carcinoma of the pancreas. This study aimed to explore the clinical profile of pancreatic intraepithelial neoplasia (PanIN), one of the precursors of carcinoma of the pancreas, in tropical CP and to identify the possible clinical predictors of the same. A retrospective study was done enrolling patients who underwent either Frey's procedure or pancreatic resection for symptomatic CP, between January 2008 and December 2012. They were grouped into PanIN positive and PanIN negative based on histopathology. Their clinical and laboratory parameters were compared statistically to identify the predictors of the PanIN status. Sixty-two tropical CP patients who had Frey's procedure or pancreatic resection were enrolled into this study; 38.7 % cases showed PanIN changes and 61.29 % had no PanIN changes. Majority of the clinical and laboratory parameters were found comparable between the two groups except obstructive jaundice and CA 19-9 value >137.5 which were there in 54.2 % of PanIN-positive cases. On histopathology, 54.83 % cases were benign CP and 45.16 % were malignant CP. Among the benign CP, 33.3 % were PanIN positive and among those with malignancy 66.7 % were PanIN positive. Low-grade PanINs were seen in 73 % cases of benign CP and in 26.1 % of malignancy whereas high-grade PanIN-3 lesions were seen exclusively in patients with malignancy. High-grade PanIN-3 lesions showed significant association with pancreatic malignancy. Obstructive jaundice and CA 19-9 ≥ 137.5 could predict PanIN positivity.

The biological features of PanIN initiated from oncogenic Kras mutation in genetically engineered mouse models

Pancreatic intraepithelial neoplasia (PanIN) is the most common premalignant lesion of the pancreas. Further understanding of the biological behavior and molecular genetic alterations in the stepwise progression of PanINs is necessary toward the development of pancreatic ductal adenocarcinoma (PDAC) interventions. In this study, we analyzed the morphological characteristics, molecular alterations, and biological behavior of pancreatic wild-type and neoplasia tissues, including analysis of PanIN cell line SH-PAN (isolated from Pdx-1-Cre; LSL-KrasG12D/+ mouse) and PDAC cell line DT-PCa (isolated from Pdx1-Cre; LSL-KrasG12D/+; LSL-Tp53R172H/+ mouse). Results show that KrasG12D induces ductal lesion PanINs. Increased expression of EGFR, Her-2/Neu, p-MAPK and β-Catenin was observed in low-grade PanINs. Tp53 was not expressed in wild-type and low-grade PanINs, however, increased expression was observed in high-grade PanINs. Furthermore, SH-PAN cells did not exhibit any colony formation and showed significantly lower migration and invasion ability compared with DT-PCa cells. Notably, we first found PPP2R2A (protein phosphatase 2, regulatory subunit B, alpha) expression was significantly higher in SH-PAN cells than DT-PCa cells, and was high in 96 of 172 peritumoral normal human pancreatic tissues and 20 of 36 human low- or middle-grade PanIN tissues, whereas, was weak or negligible in 12 of 20 human high-grade PanIN tissues and 124 of 172 human PDAC tissues post-operation. The expression of PPP2R2A appears to be correlated with clinical survival. Taken together, KrasG12D – driven PanIN showed the tumorigenic ability, however, did not undergo a malignant transformation, and decreased expression of PPP2R2A in PDACs may provided a new target for pancreatic carcinoma intervention.

Inhibition of pancreatic cancer stem cell characteristics and tumor growth by Gli inhibitor GANT61

s / Pancreatology 13 (2013) e1–e94 e11 mTOR signaling in PaSC. Further, conditioned medium from PanIN cells isolated from KRAS mice enhanced fibrogenic responses in PaSC. Histological analysis of tissue microarrays of patient-matched normal human pancreas parenchyma and PanIN lesions showed profuse active PaSC surrounding both low-grade and advanced PanINs, and acini in adjacent normal parenchymal sections. Conclusions: Our data support a key role for Akt/mTOR signaling in PaSC fibrogenic responses, and indicate that interplay between PanINs and PaSC provide the mechanistic underpinnings of environmental risk factor promotion of tumor progression.

Genome-Wide Somatic Copy Number Alterations in Low-Grade PanINs and IPMNs from Individuals with a Family History of Pancreatic Cancer

Characterizing the earliest chromosomal alterations of pancreatic precursor neoplasms from individuals with a familial aggregation of pancreatic cancer may provide clues as to the loci of pancreatic cancer susceptibility genes. We used Illumina 370/660K SNP arrays to conduct genome-wide copy number analysis in 60 benign neoplasms [58 mostly low-grade pancreatic intraepithelial neoplasias (PanIN) and intraductal papillary mucinous neoplasms (IPMN) and two pancreatic neuroendocrine tumors (PNET)] and matched normal tissues from 16 individuals with a family history of pancreatic cancer. PanINs and IPMNs were analyzed for KRAS codon 12/13 mutations. Of 40 benign neoplasms with adequate SNP calls and allele ratios, somatic chromosomal copy number changes were identifiable in only nine lesions, including eight of the 38 PanIN/IPMNs (two of which had identical alterations) and one of the two PNETs. Only two precursor lesions had more than one somatic copy number alteration. In contrast, the overwhelming majority (∼95%) of PanINs harbored KRAS mutations. The chromosomal alterations identified included nine chromosomal arms affected by chromosomal loss and two by chromosomal gain. Copy number loss spanning 9p21.3 was identified in three precursor lesions; two precursors had chromosomal losses affecting 6q and 17p. Low- and intermediate-grade PanINs and IPMNs from patients with a family history of pancreatic cancer harbor few if any somatic chromosomal alterations. The absence of a locus of recurrent chromosomal loss in most low-grade pancreatic cancer precursor lesions supports the hypothesis that there is no one tumor suppressor gene locus consistently involved in initiating familial pancreatic neoplasia.

Expression of cancer stem cell markers in pancreatic intraepithelial neoplasias and pancreatic ductal adenocarcinomas

Cancer stem cells (CSCs) play pivotal roles in cancer growth, invasion, metastasis and recurrence. Several proteins have been reported as CSC markers for pancreatic ductal adenocarcinoma (PDAC). In the present study, we examined the correlation between pancreatic intraepithelial neoplasias (PanINs) and CSC markers including CD24, CD44, CD133, CXCR4, ESA and nestin using immunohistochemical analysis. Furthermore, we examined the roles and clinical significance of these CSC markers in PDAC. CD24-, CD44-, CXCR4-, ESA- and nestin-positive cells were detected in the following tissues, listed in order of increasing percentage: normal ducts < low-grade PanINs < high-grade PanINs < PDACs. CD133 did not increase according to the malignancy grade. In PDAC, cells positive for each of the following CSC markers were detected, listed according to increasing percentage: nestin < CD133 < CD44 < CD24 < CXCR4 < ESA. CXCR4 and ESA expression correlated with well-differentiated PDAC. Venous invasion was positively associated with CD133 and inversely associated with ESA. CSC marker expression levels detected in PDAC cell lines using flow cytometry showed lowest expression of CD133 and highest of CD44, differing from the results obtained using immunohistochemistry. In two PDAC subtypes, adenosquamous carcinoma and anaplastic carcinoma, ESA was expressed more abundantly in adenocarcinoma components, whereas CD44 and nestin showed high expression in anaplastic components. Together, these results suggest that most CSC markers correlate with pancreatic carcinogenesis through the PanIN-to-PDAC sequence. Each CSC marker was related in a different manner with proliferation, differentiation, invasiveness or tissue type of PDAC.

Very high CEA level in a large pancreatic cyst: Is it a surgical indication by itself?

BackgroundPancreatic mucinous cystic lesions might develop malignancy if untreated, or could harbor malignancy at the time of the diagnosis. Many reports stated that cyst fluid carcinoembryonic antigen is an accurate diagnostic marker of pancreatic mucinous cysts. MethodsA man with a incidental pancretic cystic lesion of 35 mm in diameter was admitted to our Department. CT and EUS did not reveal solid components, main duct was not dilated and cyst fluid CEA was very high (1445 ng/ml). ResultsThe patient underwent a pancreatoduodenectomy and the surgical specimen showed a pseudocyst with columnar mucinous epithelium, consistent with low-grade PanIN. ConclusionsIs it possible that the mucinous epithelium of panIN was responsible for the unexpectedly high CEA value?Clinicians should be aware of the usefulness of the CEA level in cystic fluid but even a very high CEA value should not be considered by itself to be evidence of a mucinous lesion.

Understanding Metastasis in Pancreatic Cancer: A Call for New Clinical Approaches

Although metastasis is a major cause of morbidity and mortality in patients with pancreatic cancer, the requisite events are currently unknown. In this issue of Cell, Haeno etal. and Rhim etal. propose that metastasis occurs much earlier than previously anticipated, with clear implications for improving patient care.

Loss of Heterozygosity Status of D9S105 Marker Is Associated with Downregulation of Krüppel-Like Factor 4 Expression in Pancreatic Ductal Adenocarcinoma and Pancreatic Intraepithelial Lesions

The transcription factor Krüppel-like factor 4 (KLF4) may act both as an oncogene and a tumor suppressor in a tissue-dependent manner, and further studies on its role in pancreatic ductal adenocarcinoma (PDAC) progression and clinical outcome are warranted. Therefore, we investigated the loss of heterozygosity (LOH) in the 9q22.3–32 region and loss of KFL4 gene expression in epithelial cells from 35 PDAC, 6 pancreatic intraductal neoplasias (PanINs) and 6 normal ducts, isolated by laser microdissection, as well as their correlation with overall survival (OS) in patients treated with gemcitabine in the adjuvant setting. LOH was evaluated with 4 microsatellite markers and in situ hybridization, while KLF4 expression was studied by reverse transcription-PCR and immunohistochemistry. LOH in at least 1 locus was observed in 25 of 35 PDAC cases and in 5 of 6 PanINs, respectively. In particular, the loss of the D9S105 marker was present in 46.9% of PDAC and 83.3% of PanlNs, becoming the most deleted marker, while no LOH in D9S105 was observed in normal Wir-sung pancreatic duct. Lack of KLF4 mRNA expression was significantly associated with: (1) genomic deletion flanking KLF4 in PDAC and in PanINs (with LOH of D9S105), (2) low-grade PDAC-associated PanIN, (3) lack of KLF4 protein expression, and (4) shorter OS. These results strongly suggest a relationship between D9S105 deletion and downregulation of KLF4 gene expression as an early event in PDAC progression, as well as a possible role of KLF4 as a prognostic biomarker in gemcitabine-treated patients.

Five years of prospective screening of high-risk individuals from families with familial pancreatic cancer

Objective:Familial pancreatic cancer (FPC) accounts for approximately 3% of all pancreatic cancer (PC) cases. It has been suggested that high-risk individuals (HRIs) should be offered a screening programme.Aim:To evaluate the...

Pancreatic Intraepithelial Neoplasia Revisited and Updated

Most pancreatic neoplasms are classified as ductal adenocarcinoma because they show a ductal phenotype, making a ductal origin very likely. The duct lesions that may give rise to pancreatic ductal adenocarcinoma have been called pancreatic intraepithelial neoplasia (PanIN). A classification system for these lesions distinguishes between three grades of PanIN. Molecular studies revealed that PanIN-2 and PanIN-3 lesions represent a distinct step towards invasive carcinoma. While high-grade PanINs are extremely rare in the normal pancreas, low-grade PanINs are common in individuals older than 40 years and may be associated with lobular fibrosis and intraductal papillary mucinous neoplasms of the gastric type. This disease spectrum has also been described in members of kindreds with familial pancreatic cancer. The natural history and cause of PanINs are unknown. As PanIN-1 lesions entail little risk, while PanIN-3 lesions are high-risk lesions, it would be of interest to target PanIN-2 lesions, which can be regarded as the starting point of progressive neoplastic changes that lead to invasive pancreatic ductal adenocarcinoma. Global gene expression analysis identified several differentially expressed genes which show enhanced expression in PanINs and may be used as potential biomarkers to facilitate diagnosis and therapy.

Expression of COX-2 is associated with accumulation of p53 in pancreatic cancer: analysis of COX-2 and p53 expression in premalignant and malignant ductal pancreatic lesions

Cyclooxygenase-2 (COX-2) and tumor suppressor p53 are molecules that are linked to the oncogenesis of pancreatic cancer. COX-2 represents a key modulatory molecule in inflammation and carcinogenesis, and is known to be implicated in the positive regulation of growth and tumorigenesis. Abnormal expression of p53 is common in many human neoplasms including pancreatic cancer. Recent studies demonstrated functional interactions between p53 and COX-2. The p53-dependent upregulation of COX-2 was proposed to be another mechanism by which p53 could abate its own growth-inhibitory and apoptotic effects. In this study, we immunohistochemically analyzed the expression of COX-2 and p53 in 95 pancreatic resection specimens [adenocarcinomas, 95 lesions; pancreatic intraepithelial neoplasias (PanINs), 155; normal ducts, 70]. The expression of COX-2 increased progressively with the grade of ductal lesions (P<0.00001). A statistically significant difference of COX-2 expression between normal ducts and low-grade PanINs was revealed (P=0.0042). COX-2 overexpression was demonstrated in 82 PanINs (52.9%), and in 76 adenocarcinomas (80%). No significant correlation between the grade of adenocarcinoma and COX-2 expression was revealed (P=0.2). The expression of p53 again increased progressively with the grade of lesions (P<0.00001) with a significant increase in high-grade PanINs. A correlation between COX-2 and p53 expression levels in carcinomas was revealed (P=0.0002), and an accumulation of p53 was associated with COX-2 overexpression in premalignant and malignant ductal lesions. These findings confirmed the generally accepted pancreatic cancer progression model, and supported the concept of the interactive role of COX-2 and p53 in pancreatic cancer carcinogenesis, which offers opportunities for targeted therapy and chemoprevention of pancreatic cancer using COX-2 inhibitors.

Current understanding of precursors to pancreatic cancer

Precursors to pancreatic cancer have been investigated for a century. Previous studies have revealed three distinct precursors, i.e. mucinous cystic neoplasm (MCN), intraductal papillary mucinous neoplasm (IPMN), and pancreatic intraepithelial neoplasia (PanIN), harboring identical or similar genetic alterations as does invasive pancreatic carcinoma. The current understanding of precursors to pancreatic cancer can be illustrated by progressive pathways from noninvasive MCN, IPMN, and PanIN toward invasive carcinoma. MCNs consist of ovarian-type stroma and epithelial lining with varying grades of atypia, and are occasionally associated with invasive adenocarcinoma. The epithelium of noninvasive IPMNs shows a variety of different directions of differentiation, including gastric, intestinal, pancreatobiliary (PB), and oncocytic types. IPMNs can also harbor varying grades of architectural and cytologic atypia. IPMNs confined to branch ducts are mostly the gastric type, and IPMNs involving the main ducts are often intestinal type, while PB and oncocytic types are rare. Small (<1 cm) IPMNs of the gastric type are not always morphologically distinguishable from low-grade PanINs. Mucin expression profiles suggest intestinal-type IPMNs progress to mucinous noncystic (colloid) carcinoma, while PB-type IPMNs progress toward ductal adenocarcinoma. It is a well-described paradigm that PanIN lesions progress toward ductal adenocarcinoma through step-wise genetic alterations. The activation of Hedgehog and Notch signaling pathways in PanIN lesions as well as in pancreatic adenocarcinoma suggest that developmental pathways may be disregulated during carcinogenesis of the pancreas. Further study is needed to elucidate the pathways from precursors toward invasive carcinoma of the pancreas.

Tenascin C and annexin II expression in the process of pancreatic carcinogenesis

Tenascin C (TNC) is a component of the provisional extracellular matrix (ECM) that characterizes solid tumours. Cell surface annexin II is a high-affinity receptor for large TNC splice variants. The aim of this study was to analyse whether TNC and annexin II play a role in the development of pancreatic ductal adenocarcinoma (PDAC). PDAC is characterized by a rich ECM populated by pancreatic stellate cells, which play a crucial role in pancreatic desmoplasia. The mRNA and protein levels of TNC and of annexin II were analysed in pancreatic tissues by DNA array, quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) and immunohistochemistry. TNC large splice variants were detected by RT-PCR. Enzyme linked immunosorbent assay (ELISA) was used to measure TNC levels in serum and culture supernatants. TNC and annexin II mRNA levels were significantly higher in pancreatic cancer tissues than in the normal pancreas. TNC expression was detected with increased frequency in the progression from PanIN-1 lesions to PDAC, and a parallel switch from cytoplasmic to cell surface expression of annexin II was observed. Large TNC transcripts were found in pancreatic cancer and in chronic pancreatitis, but not in the normal pancreas. TNC expression was demonstrated in pancreatic stellate cells, where it could be induced by tumour necrosis factor alpha (TNFalpha), transforming growth factor beta1 (TGF-beta1) and by cancer cell supernatants supplemented with TGF-beta1. In conclusion, the expression of TNC and cell surface annexin II increases in the progression from low-grade PanIN lesions to pancreatic cancer. Pancreatic stellate cells are identified as a source of TNC in pancreatic tissues, possibly under the influence of soluble factors released by the tumour cells.

Epithelial-cadherin and beta-catenin expression changes in pancreatic intraepithelial neoplasia.

Cadherins and associated catenins are important mediators of epithelial cell-cell adhesion, as well as the Wnt-signaling pathway. Significant changes in their expression or structure have been implicated in malignancy. This study aimed to investigate the epithelial-cadherin (E-cadherin) and beta-catenin expression changes during multistage, pancreatic ductal carcinogenesis. Ninety-four Whipple resection specimens were retrieved from the surgical pathology files of the University Health Network (Toronto, Canada), from which tissue microarray blocks containing 36 pancreatic ductal adenocarcinomas, 34 PanIN-1A lesions, 28 PanIN-1B lesions, 27 PanIN-2 lesions, 16 PanIN-3 lesions, and 32 normal ducts were constructed. The E-cadherin, beta-catenin, and the phosphorylated glycogen synthase kinase-3beta of the Wnt/beta-catenin pathway were immunohistochemically evaluated in these duct/PanIN lesions. There was marked increase in the cytoplasmic E-cadherin expression in PanIN lesions (P < 0.0001) and adenocarcinoma (P = 0.005) compared with normal pancreatic ducts. In contrast, reduced/loss of E-cadherin membranous expression was also significant in ductal adenocarcinoma compared with both the PanIN lesions (P < 0.0001) and normal ducts (P = 0.05). The beta-catenin expression showed significantly more frequent aberrant nuclear localization in high-grade PanIN lesions, particularly PanIN2 and in adenocarcinoma compared with normal ducts or low grade PanIN lesions (P < 0.0001). However, there was a lack of correlation between phospho(Ser9)-glycogen synthase kinase-3beta cytoplasmic expression and beta-catenin aberrant nuclear expression (P = 0.07). Aberration in the expression of E-cadherin and its associated beta-catenin is evident in pre-invasive (PanIN) neoplastic pancreatic duct cells, suggesting involvement of pathways leading to beta-catenin stabilization during pancreatic duct cell carcinogenesis.